This invention relates generally to the determination of the amount of creatinine in urine or other bodily fluids. More particularly, the invention relates to an improvement in creatinine assays in which copper complexes with creatinine and then acts as a oxidant for a indicator, for example one in which a color change provides an indication of the creatinine concentration.
Creatinine is the end metabolite when creatine becomes creatine phosphate and is used as an energy source for muscle contraction. The creatinine product is filtered in the kidneys and excreted into the urine. The amount of the creatinine in the urine provides an indicator useful for diagnosing muscle diseases or kidney diseases. Various tests have been used for measuring the concentration of creatinine, such as those discussed in the patents related to the present invention and discussed below.
In a series of US Patents, Miles, Inc. and later the Bayer Corporation, disclosed methods of analysis that employed copper complexes serving as pseudoperoxidases for oxidizing redox indicators such as 3,3′,5,5′-tetramethylbenezidine (TMB). The earliest of these was U.S. Pat. No. 5,173,431 in which a copper/protein complex was used and an ionizable phosphate was included in the system to prevent interference by components in the biological fluid being tested. In U.S. Pat. No. 5,374,561, it was shown that a complex of CuII with creatinine was capable of oxidizing a redox indicator to provide a measure of the amount of creatinine present in a urine sample. The sample was brought into contact with a test strip containing cupric ions, a hydroperoxide, and a redox indicator. A set of reactions was postulated in which the creatinine in the sample was complexed with the cupric ions and the complex oxidized the indicator to a colored form. The reduced copper/creatinine complex was reoxidized to the cupric form by the hydroperoxide. Improvements in the method of the '561 patent were disclosed in U.S. Pat. Nos. 5,733,787; 6,001,656; and 6,210,971. The improved methods reduce interference of components in the sample and extend the useful shelf life of the test strips in order that more accurate results can be obtained.
Other methods of detecting of creatinine have been established. Some of these involve the use of fluorescent materials to detect the presence of creatinine, rather than by developing a color. One such method is disclosed in U.S. Pat. No. 5,958,786 in which a fluorescent dye, e.g. acridine orange, was used to detect ammonia produced by the reaction of creatinine with the enzyme creatinine deiminase. In U.S. Pat. No. 5,527,708 creatinine was reacted with 3,5-dinitrobenzoate to produce a fluorescent material. In neither of these methods was a copper/creatinine complex formed.
While the use of copper/creatinine complexes has been shown to be successful in measuring creatinine in urine samples, further improvement is, as always, sought by those skilled in this art. Since current methods use enzymes, which are more costly and unstable, or are colorometric and are optically interfered in serum. It would be preferred to use a less costly method or, if the application is in serum, use one that is free from optical interferences.
Thus, the present inventors continued investigation of creatinine measurement and discovered the new method which will be described in detail below. In general, the colorimetric indicator is replaced by a fluorescent indicator, which is especially useful in measurement of creatinine in blood and other bodily fluids.
Fluorescent indicators have been used for analytes that have an oxidase specific toward them such as cholesterol or glucose. In one example, 4-(1-methylhydrazino)-7-nitrobenzooxadiazole (MNBDH) was used as a fluorogenic peroxidase indicator to produce fluorescence when oxidized by hydrogen peroxide, which had been generated by the reaction of glucose with glucose oxidase. See Meyer, J.; Buldt, A.; Vogel, M. Karst, U., Angew. Chem. Int. Ed. 2000, 39(8) 14 53 and Ger. Patent 199 32 380 A 1, 2000.
Although measurement of the creatinine concentration in urine has been the focus of the patents discussed above, creatinine also is found in blood. Serum and plasma concentrations of creatinine elevate in extensive renal disease, especially from decreased glomular filtration.
A single random serum creatinine measurement may be used as an indicator of renal failure. However, a more sensitive test for measuring glomular filtration is the creatinine clearance test. In this test, the serum creatinine concentration is used in conjunction with the measurement of creatinine in the precisely timed (usually 24 hr) urine sample to estimate the Glomular Filtration Rate (GFR). Creatinine clearance is used to follow the clinical course and therapeutic response in acute glomerulonephritis, to discriminate between acute glomular disease vs. chronic structural damage and as a measurement of overall renal function. Presently, creatinine in blood is measured by variations of the Jaffe method, which is sensitive to blood interferants such as glucose, ascorbate, uric acid, keto acids, pyruvate, proteins cephalosporins drugs and background signal. Alternatively, enzymatic methods are also used, which are expensive and suffer from enzyme instabilities. Thus, extension of the specific yet less complex creatinine complex method would constitute an improvement in the art of creatinine detection. The present inventors have found the new method which is described in detail below.